This Project This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background
ZBTB7A (Zinc Finger and BTB Domain containing 7A), also known as the Pokemon gene, is part of the POK family, a family of tumor repressors (8). It is located on 19p13.3, and is comprised of 584 amino acids (11). Its primary function is the suppression of the p14ARF tumor suppressor gene (2). ZBTB7A prevents a common trait of cancer, accelerated glycolysis, by binding to the promoter and preventing the transcription of important glycolytic genes, such as GLUT3, PFKP, and PKM (7). Therefore, ZBTB7A is a proto-oncogene, as portrayed through the lack of this gene in many human tumors and the increased dependency of ZBTB7A-lacking cancer cells on glycolysis (7). ZBTB7A is also known to perform a number of other biological functions. It is known as a binding factor to a specific part of the human immunodeficiency virus. In addition, it is a part of processes, such as osetoclastogenesis, chondrogenesis, and adipogenesis, while playing a pivotal role in cell tumorigenesis (11).
Picture 1. Image of Structure of ZBTB7A protein.
This image is from Wikipedia.
Methods
The sequence for the assigned protein was obtained from the Ensembl protein database in a FASTA format. Through a Blast in the whaleshark.georgiaaquarium.org Galaxy server, the human protein sequence (ENSP00000323670) was queried against the whale shark protein database. The five top protein hits were then crosschecked as queries in BLASTS against the human protein database in NCBI to determine which were the closest matches. The alignment lengths, E-Values, query coverage, and identity were used to determine the closest matches. ZBTB7A/LRF predicted orthologs were obtained from Bottle-nose dolphins, elephant sharks, zebra fish, and dogs. The Human FASTA sequence was used as the query when performing the BLASTS for the organisms mentioned in NCBI. The best hit for each organism and each whale shark protein BLAST were used to create a phylogenetic tree through ClustalW.
Searching the Whale Shark Genome
Table 1. Possible Whale Shark ZBTB7A/LRF Matches
Table 1 shows the five most compatible protein matches from the Whale shark genome upon comparison to the human ZBTB7A/LRF gene.
Proteins were selected based on low e-values and high % identities.
In order to determine whether the ZBTB7A/LRF gene existed in the Whale shark, the protein's FASTA was uploaded to Georgia Aquarium's Galaxy website and was compared to the Whale shark's genome. Upon analyzing the table, we are able to conclude that although the protein matches from the Whale shark genome aren't exact, they are relatively close to the human protein.
Protein Domains
Figure 1. BLAST output from all species + human ZBTB7A/LRF gene.
The best hit ZBTB7A/LRF gene results for all BLASTs between the ZBTB7A/LRF gene and the protein sequences of other species reveals that each contains a BTB superfamily and C0G5028 multi-domains. The BTB superfamily is known to cell functions, with transcriptional regulation being one of them (10).
Orthologues
The human ZBTB7A/LRF protein sequence (ESPN00000323670) was used as query in NCBI BLAST searches against individual species' protein databases. Table 2 displays the best matches with each species when blasted against the human ZBTB7A/LRF sequence. Interpreting the data, we can conclude that the ZBTB7A/LRF protein was found in all tested species except the yeast and the fruit fly; only the mammals seem to carry this gene. Therefore, it is safe to assume there was a divergence between mammals and non-mammals.
Table 2. Best Protein Hits with human ZBTB7A/LRF protein sequence. The ZBTB7A/LRF gene was used and protein BLASTs were performed against species individually. Displayed are the species, followed by the closest protein hit, its max score, total score, query cover, e-value, % identity, and accession.
Phylogeny
The best protein hits for each respective species plus the five top whale shark genome hits found using the ZBTB7A/LRF gene as query was used to create a phylogenetic tree. Firstly, we can conclude that the five whale shark proteins are relatively similar (Figure 2). Also, the fact that the fruit fly and yeast are found to be similar with the whale shark genomes is was unexpected. However, this information indicates that whale sharks may have BTB factors that may not be directly from ZBTB7A/LRF. The protein found in the whale shark genome may also be a more ancestral form of ZBTB7A/LRF considering its position on the phylogenetic tree (Figure 2).
Figure 2. Phylogenetic tree of ZBTB7A/LRF gene best hits. The best hits for each respective species, and the five best hits from the whale shark genome were imputed into the ClustalW2 program, creating a phylogenetic tree revealing the relations between the different proteins and branch lengths determine approximately during what time periods the divergences took place.
Conclusions
Although the exact ZBTB7A/LRF gene could not be identified in the whale shark genome, it can be assumed that a divergence occurred early in the phylogenetic tree, causing for different variants of the BTB superfamily. Sharks are known to have existed for about 450 millions years (1). This strongly suggests that a divergence occurred and the ZBTB7A/LRF ortholog exists in a more ancestral version of the BTB protein. Because our research revealed the presence of a similar protein sequences in the whale shark genome despite the lack of the identical protein (Table 1), further research can be done to discover the exact protein that has commonalities with the human protein ZBTB7A/LRF gene. In other words, by researching deeper into the phylogenetic tree, the ancestral gene can be discovered. In addition, because it was revealed that yeast and the fruit-fly, or the non-mammalian species, was found to be of similar status as the whale shark (Figure 2), further research delving into these species would be of use as well in determining the whale shark's parallel to the ZBTB7A/LRF gene.
References
"450 Million Years of Sharks." Shark Savers ::. N.p., n.d. Web. 1 Apr. 2015.
Apostolopoulou, K., Pateras, I., Evangelou, K., Tsantoulis, P., Liontos, M., Kittas, C., Tiniakos, D., Kotsinas, A., Cordon-Cardo, C. and Gorgoulis, V. (2007), Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer. J. Pathol., 213: 294–302. doi: 10.1002/path.2222
Liu, Xue-Song et al. “ZBTB7A Acts as a Tumor Suppressor through the Transcriptional Repression of Glycolysis.” Genes & Development 28.17 (2014): 1917–1928. PMC. Web.
Maeda, Takahiro, Robin M. Hobbs, Taha Merghoub, Ilhem Guernah, Arthur Zelent, Carlos Cordon-Cardo, Julie Teruya-Feldstein, and Pier Paolo Pandolfi. "Role of the Proto-oncogene Pokemon in Cellular Transformation and ARF Repression." Nature 433.7023 (2005): 278-85. Web.
Stogios, Peter J., Gregory S. Downs, Jimmy JS Jauhal, Sukhjeen K. Nandra, and Gilbert G. Prive. "Sequence and Structural Analysis of BTB Domain Proteins." Genome Biology 6.10 (2005): n. pag. Web. <http://genomebiology.com/2005/6/10/r82>.
Wang, Guocan et al. “Lrf Suppresses Prostate Cancer through Repression of a Sox9-Dependent Pathway for Cellular Senescence Bypass and Tumor Invasion.” Nature genetics 45.7 (2013): 739–746. PMC. Web.
Zhao, Zhi-Hong, Sheng-Fa Wang, Liang Yu, Ju Wang, Hao Chang, Wei-Li Yan, Jian Zhang, and Kai Fu. "Overexpression of Pokemon in Non-small Cell Lung Cancer and Foreshowing Tumor Biological Behavior as Well as Clinical Results." Lung Cancer 62.1 (2008): 113-19. Web.
This ProjectThis web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.
Background
ZBTB7A (Zinc Finger and BTB Domain containing 7A), also known as the Pokemon gene, is part of the POK family, a family of tumor repressors (8). It is located on 19p13.3, and is comprised of 584 amino acids (11).
Its primary function is the suppression of the p14ARF tumor suppressor gene (2). ZBTB7A prevents a common trait of cancer, accelerated glycolysis, by binding to the promoter and preventing the transcription of important glycolytic genes, such as GLUT3, PFKP, and PKM (7). Therefore, ZBTB7A is a proto-oncogene, as portrayed through the lack of this gene in many human tumors and the increased dependency of ZBTB7A-lacking cancer cells on glycolysis (7).
ZBTB7A is also known to perform a number of other biological functions. It is known as a binding factor to a specific part of the human immunodeficiency virus. In addition, it is a part of processes, such as osetoclastogenesis, chondrogenesis, and adipogenesis, while playing a pivotal role in cell tumorigenesis (11).
Picture 1. Image of Structure of ZBTB7A protein.
This image is from Wikipedia.
Methods
The sequence for the assigned protein was obtained from the Ensembl protein database in a FASTA format. Through a Blast in the whaleshark.georgiaaquarium.org Galaxy server, the human protein sequence (ENSP00000323670) was queried against the whale shark protein database. The five top protein hits were then crosschecked as queries in BLASTS against the human protein database in NCBI to determine which were the closest matches. The alignment lengths, E-Values, query coverage, and identity were used to determine the closest matches. ZBTB7A/LRF predicted orthologs were obtained from Bottle-nose dolphins, elephant sharks, zebra fish, and dogs. The Human FASTA sequence was used as the query when performing the BLASTS for the organisms mentioned in NCBI. The best hit for each organism and each whale shark protein BLAST were used to create a phylogenetic tree through ClustalW.
Searching the Whale Shark Genome
Table 1. Possible Whale Shark ZBTB7A/LRF Matches
Table 1 shows the five most compatible protein matches from the Whale shark genome upon comparison to the human ZBTB7A/LRF gene.
Proteins were selected based on low e-values and high % identities.
In order to determine whether the ZBTB7A/LRF gene existed in the Whale shark, the protein's FASTA was uploaded to Georgia Aquarium's Galaxy website and was compared to the Whale shark's genome. Upon analyzing the table, we are able to conclude that although the protein matches from the Whale shark genome aren't exact, they are relatively close to the human protein.
Protein Domains
Figure 1. BLAST output from all species + human ZBTB7A/LRF gene.
The best hit ZBTB7A/LRF gene results for all BLASTs between the ZBTB7A/LRF gene and the protein sequences of other species reveals that each contains a BTB superfamily and C0G5028 multi-domains. The BTB superfamily is known to cell functions, with transcriptional regulation being one of them (10).
Orthologues
The human ZBTB7A/LRF protein sequence (ESPN00000323670) was used as query in NCBI BLAST searches against individual species' protein databases. Table 2 displays the best matches with each species when blasted against the human ZBTB7A/LRF sequence. Interpreting the data, we can conclude that the ZBTB7A/LRF protein was found in all tested species except the yeast and the fruit fly; only the mammals seem to carry this gene. Therefore, it is safe to assume there was a divergence between mammals and non-mammals.
Table 2. Best Protein Hits with human ZBTB7A/LRF protein sequence. The ZBTB7A/LRF gene was used and protein BLASTs were performed against species individually. Displayed are the species, followed by the closest protein hit, its max score, total score, query cover, e-value, % identity, and accession.
Phylogeny
The best protein hits for each respective species plus the five top whale shark genome hits found using the ZBTB7A/LRF gene as query was used to create a phylogenetic tree. Firstly, we can conclude that the five whale shark proteins are relatively similar (Figure 2). Also, the fact that the fruit fly and yeast are found to be similar with the whale shark genomes is was unexpected. However, this information indicates that whale sharks may have BTB factors that may not be directly from ZBTB7A/LRF. The protein found in the whale shark genome may also be a more ancestral form of ZBTB7A/LRF considering its position on the phylogenetic tree (Figure 2).
Figure 2. Phylogenetic tree of ZBTB7A/LRF gene best hits. The best hits for each respective species, and the five best hits from the whale shark genome were imputed into the ClustalW2 program, creating a phylogenetic tree revealing the relations between the different proteins and branch lengths determine approximately during what time periods the divergences took place.
Conclusions
Although the exact ZBTB7A/LRF gene could not be identified in the whale shark genome, it can be assumed that a divergence occurred early in the phylogenetic tree, causing for different variants of the BTB superfamily. Sharks are known to have existed for about 450 millions years (1). This strongly suggests that a divergence occurred and the ZBTB7A/LRF ortholog exists in a more ancestral version of the BTB protein. Because our research revealed the presence of a similar protein sequences in the whale shark genome despite the lack of the identical protein (Table 1), further research can be done to discover the exact protein that has commonalities with the human protein ZBTB7A/LRF gene. In other words, by researching deeper into the phylogenetic tree, the ancestral gene can be discovered. In addition, because it was revealed that yeast and the fruit-fly, or the non-mammalian species, was found to be of similar status as the whale shark (Figure 2), further research delving into these species would be of use as well in determining the whale shark's parallel to the ZBTB7A/LRF gene.
References